Human Immunodeficiency Virus type-1 (HIV-1) reverse transcriptase (RT) is a highly error-prone enzyme that is thought to be responsible for the generation of viral genetic diversity. This in turn has been suggested to be essential to the virus' ability to evade host immune responses, and to establish a state of persistent, productive infection in the host. Our hypothesis is that viruses with enhanced replicational fidelity will generate fewer mutants than wild-type viruses, and that such "high- fidelity" viruses may be incapable of escaping from host immune pressure, due to their inability to spawn highly diverse quasispecies. If correct, this hypothesis could have important implications for the design of live-attenuated HIV vaccines. This application represents a proof-of-concept study that is designed to test whether a "high fidelity" primate lentivirus is indeed attenuated in terms of the generation of new mutants and escape from immune pressure within a nonhuman primate host. The Simian Immunodeficiency Virus (SIV)/macaque model system will be used for these experiments. In earlier studies, we have created HIV-1 and SIV RT mutants with increased replicational fidelity (approx. 10-fold greater than wild-type). Similar methods will be used to create several high-fidelity mutants of SIVmac239 RT. After biochemical characterization, these RT mutants will be substituted into an intact SIVmac239 molecular clone, and virus stocks will be generated (SIVmac239-hifi). The replicational fitness and fidelity of these viruses will be tested in vitro, and a clone with wild-type replicational fitness but enhanced replicational fidelity will then be selected for in vivo studies. This virus will be inoculated into rhesus macaques (both Mamu-A*01 positive and Mamu-A*01 negative animals), and the development of virus-specific, Mamu-A*01 restricted CTL responses will be examined using MHC:peptide tetramers. Plasma virus load will be measured at selected time points following infection, and viral genetic diversity will be analyzed at several loci, including a Mamu-A*01 restricted Tat CTL epitope that has previously been to shown to undergo very rapid replacement in SIVmac239-infected, Mamu-A*01 positive rhesus macaques, but not in infected Mamu-A*01 negative animals. These experiments are expected to provide insight as to the potential utility of using high fidelity RT mutants to improve the safety of live-attenuated HIV vaccines.